Manipulation of molecules relative to living cells, such as drug delivery into cells or isolation of cell components, including, for example, integral membrane proteins, has been limited by the nature of living cell membranes. The integrity of living cells is maintained by cell membranes which include a bilayer of phospholipids. Phospholipids are comprised of a hydrophilic lipid head group and one or more hydrophobic fatty acyl chains They are characterized as being amphipathic because they have both a hydrophilic component and a hydrophobic component. A cell membrane lipid bilayer includes substantially aligned hydrophobic fatty acyl chains of the phospholipids and two layers of the hydrophilic lipid heads which shield the fatty acyl chains from a medium in which the cell is disposed.
The amphipathic nature of phospholipids causes cell membranes to be substantially impermeable to ions and, with the exception of water, most polar molecules Migration of nutrients and waste products across cell membranes are generally mediated by specific transport mechanisms of integral membrane proteins. Integral membrane proteins typically span cell membranes and include hydrophobic side chains at one portion of the protein surface and hydrophilic side chains at another portion of the protein surface. However, delivery of water-soluble molecules, such as many drugs, into a cell through a cell membrane is limited because the hydrophobic fatty acyl layer between the two hydrophilic lipid head group layers substantially prevents migration of the molecule from an aqueous medium, in which the cell is disposed, across the cell membrane and into the cell.
Attempts to improve delivery of drugs and other molecules across cell membranes have included, for example, modification of the molecule, encapsulation of the molecule in vesicles which are tailored for uptake into specific types of cells and attachment to other molecules which are actively transported across cell membranes. However, most methods of delivery involve either adulteration of the composition to which the cell is exposed or limiting the effectiveness of the molecule, such as in the case of delivering a drug, by changing the structure of the molecule.
The amphipathic nature of lipid bilayers and integral proteins also causes manipulation of cell membranes and isolation of integral proteins to be difficult. For example, isolation of cell components, such as integral membrane proteins, often requires that the cell component be solubilized. Integral membrane proteins are typically isolated by exposing cells which include the proteins to a medium having a surfactant disposed therein. The surfactant is present in an amount sufficient to exceed a critical micelle concentration, or CMC, whereby micelles are formed. Simultaneously, the cell membranes typically dissolve and the integral membrane proteins of the cells become incorporated into the micelles of the surfactant as the micelles form, thereby solubilizing the integral membrane proteins. Hydrophobic portions of the surfactant molecules cluster around the hydrophobic portion of the integral membrane protein while the hydrophilic portions of the surfactant molecules are exposed to the surrounding aqueous medium.
However, the solubilized membrane proteins are contained in protein-detergent micelles of heterogeneous size and shape. These heterogeneous populations are difficult to crystallize, and separation of individual proteins is often compromised. Attempts to increase the resolution of proteins separated from cell membranes generally employs solvents, such as ionic detergents or alcohols, that denature proteins, thereby reducing the utility and yield of the proteins isolated.
Therefore, a need exists for a method of allowing significantly increased migration of a molecule across a lipid bilayer which overcomes or minimizes the above-mentioned problems.